CRISPR/Cas9 and Transgene Verification of Gene Involvement in Unfolded Protein Response and Recombinant Protein Production in Barley Grain.

The use of plants as heterologous hosts to produce recombinant proteins has some intriguing advantages. There is, however, the potential of overloading the endoplasmic reticulum (ER) capacity when producing recombinant proteins in the seeds. This leads to an ER-stress condition and accumulating of unfolded proteins. The unfolded protein response (UPR) is activated to alleviate the ER-stress. With the aim to increase the yield of human epidermal growth factor (EGF) and mouse leukemia inhibitory factor (mLIF) in barley, we selected genes reported to have increased expression during ER-induced stress. The selected genes were calreticulin (CRT), protein disulfide isomerase (PDI), isopentenyl diphosphate isomerase (IPI), glutathione-s-transferase (GST), HSP70, HSP26, and HSP16.9. These were knocked out using CRISPR/Cas9 or overexpressed by conventional transgenesis. The generated homozygous barley lines were crossed with barley plants expressing EGF or mLIF and the offspring plants analyzed for EGF and mLIF protein accumulation in the mature grain. All manipulated genes had an impact on the expression of UPR genes when plantlets were subjected to tunicamycin (TN). The PDI knockout plant showed decreased protein body formation, with protein evenly distributed in the cells of the endosperm. The two genes, GST and IPI, were found to have a positive effect on recombinant protein production. mLIF expression was increased in a F2 homozygous GST knockout mutant background as compared to a F2 GST wild-type offspring. The overexpression of IPI in a F1 cross showed a significant increase in EGF expression. We demonstrate that manipulation of UPR related genes can have a positive effect on recombinant protein accumulation.

Modulation of Barley (Hordeum vulgare L.) Grain Protein Sink-Source Relations Towards Human Epidermal Growth Factor Instead of B-hordein Storage Protein.

Seeds have evolutionarily developed to store protein without immediately degrading it and constitute ideal tissues for recombinant protein storage. Unfortunately, the production of recombinant protein in seeds is compromised by low yield as compared to other heterologous expression systems. In order to improve the yield of the human epidermal growth factor (EGF) in barley, protein sink-source relations in the developing grain were modulated towards EGF instead of the barley storage protein. The EGF gene, under the control of a B-hordein and a seed-specific oat globulin promoter, was introduced by crossing EGF lines into the Risø 56 mutant deficient in B-hordein storage protein synthesis. Offspring plants were analysed for EGF and Hordein expression and for expression of the unfolded protein response (UPR) genes PDI and CRT to monitor changes in ER stress levels. EGF content was increased significantly in the mature grain of homozygous offspring and PDI and CRT gene expressions were upregulated. We demonstrate, for the first time in barley, that replacement of an abundant seed storage protein with a specific heterologous protein driven by the promoter of the removed gene can accelerate the production of a specific heterologous protein in barley grains.

Horizontal Stacking of PAPhy_a Cisgenes in Barley Is a Potent Strategy for Increasing Mature Grain Phytase Activity.

Mature grain phytase activity (MGPA) in the Triticea tribe cereals has evolved through gene duplications and neo-functionalization of the purple acid phosphatase phytase gene (PAPhy) in a common ancestor. Increased gene copy number of the PAPhy_a gene expressed during seed development has augmented the MGPA in cereals like rye and wheat. PAPhy_a phytase is highly stable and a potent enzyme in feed. However, barley only contains one HvPAPhy_a gene and the MGPA levels needs to be increased to substitute for the addition of microbial phytases to the feed. A substantial increase in MGPA for cisgenic barley was achieved with one extra homozygous HvPAPhy_a insert when the plants were grown in the greenhouse. In the current study, the stability of increased MGPA was confirmed in open field grown cisgenic barley. Furthermore, the gene dose response of phytase cisgenes from three different cisgenic barley plants were horizontally stacked. Cisgenic barley with 0, 1, 2, 3, 4, and 6 extra HvPAPhy_a inserts demonstrated a perfect positive linear correlation with the level of MGPA. The current study provides new insight into the potential of stacking of cisgenes in crops and suggests cisgene stacking as a versatile strategy for crop improvement.

Identification of transcription factor genes involved in anthocyanin biosynthesis in carrot (Daucus carota L.) using RNA-Seq.

BACKGROUND: Anthocyanins are water-soluble colored flavonoids present in multiple organs of various plant species including flowers, fruits, leaves, stems and roots. DNA-binding R2R3-MYB transcription factors, basic helix-loop-helix (bHLH) transcription factors, and WD40 repeat proteins are known to form MYB-bHLH-WD repeat (MBW) complexes, which activates the transcription of structural genes in the anthocyanin pathway. Although black cultivars of carrots (Daucus carota L.) can accumulate large quantities of anthocyanin in their storage roots, the regulatory genes responsible for their biosynthesis are not well characterized. The current study aimed to analyze global transcription profiles based on RNA sequencing (RNA-Seq), and mine MYB, bHLH and WD40 genes that may function as positive or negative regulators in the carrot anthocyanin biosynthesis pathways. RESULTS: RNA was isolated from differently colored calli, as well as tissue samples from taproots of various black carrot cultivars across the course of development, and gene expression levels of colored and non-colored tissue and callus samples were compared. The expression of 32 MYB, bHLH and WD40 genes were significantly correlated with anthocyanin content in black carrot taproot. Of those, 11 genes were consistently up- or downregulated in a purple color-specific manner across various calli and cultivar comparisons. The expression of 10 out of these 11 genes was validated using real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). CONCLUSIONS: The results of this study provide insights into regulatory genes that may be responsible for carrot anthocyanin biosynthesis, and suggest that future focus on them may help improve our overall understanding of the anthocyanin synthesis pathway.

Barley HvPAPhy_a as transgene provides high and stable phytase activities in mature barley straw and in grains.

The phytase purple acid phosphatase (HvPAPhy_a) expressed during barley seed development was evaluated as transgene for overexpression in barley. The phytase was expressed constitutively driven by the cauliflower mosaic virus 35S-promoter, and the phytase activity was measured in the mature grains, the green leaves and in the dry mature vegetative plant parts left after harvest of the grains. The T2 -generation of HvPAPhy_a transformed barley showed phytase activity increases up to 19-fold (29 000 phytase units (FTU) per kg in mature grains). Moreover, also in green leaves and mature dry straw, phytase activities were increased significantly by 110-fold (52 000 FTU/kg) and 57-fold (51 000 FTU/kg), respectively. The HvPAPhy_a-transformed barley plants with high phytase activities possess triple potential utilities for the improvement of phosphate bioavailability. First of all, the utilization of the mature grains as feed to increase the release of bio-available phosphate and minerals bound to the phytate of the grains; secondly, the utilization of the powdered straw either directly or phytase extracted hereof as a supplement to high phytate feed or food; and finally, the use of the stubble to be ploughed into the soil for mobilizing phytate-bound phosphate for plant growth.

High mature grain phytase activity in the Triticeae has evolved by duplication followed by neofunctionalization of the purple acid phosphatase phytase (PAPhy) gene.

The phytase activity in food and feedstuffs is an important nutritional parameter. Members of the Triticeae tribe accumulate purple acid phosphatase phytases (PAPhy) during grain filling. This accumulation elevates mature grain phytase activities (MGPA) up to levels between ~650 FTU/kg for barley and 6000 FTU/kg for rye. This is notably more than other cereals. For instance, rice, maize, and oat have MGPAs below 100 FTU/kg. The cloning and characterization of the PAPhy gene complement from wheat, barley, rye, einkorn, and Aegilops tauschii is reported here. The Triticeae PAPhy genes generally consist of a set of paralogues, PAPhy_a and PAPhy_b, and have been mapped to Triticeae chromosomes 5 and 3, respectively. The promoters share a conserved core but the PAPhy_a promoter have acquired a novel cis-acting regulatory element for expression during grain filling while the PAPhy_b promoter has maintained the archaic function and drives expression during germination. Brachypodium is the only sequenced Poaceae sharing the PAPhy duplication. As for the Triticeae, the duplication is reflected in a high MGPA of ~4200 FTU/kg in Brachypodium. The sequence conservation of the paralogous loci on Brachypodium chromosomes 1 and 2 does not extend beyond the PAPhy gene. The results indicate that a single-gene segmental duplication may have enabled the evolution of high MGPA by creating functional redundancy of the parent PAPhy gene. This implies that similar MGPA levels may be out of reach in breeding programs for some Poaceae, e.g. maize and rice, whereas Triticeae breeders should focus on PAPhy_a.

TAL effector nucleases induce mutations at a pre-selected location in the genome of primary barley transformants.

Transcription activator-like effector nucleases (TALENs) enable targeted mutagenesis in a variety of organisms. The primary advantage of TALENs over other sequence-specific nucleases, namely zinc finger nucleases and meganucleases, lies in their ease of assembly, reliability of function, and their broad targeting range. Here we report the assembly of several TALENs for a specific genomic locus in barley. The cleavage activity of individual TALENs was first tested in vivo using a yeast-based, single-strand annealing assay. The most efficient TALEN was then selected for barley transformation. Analysis of the resulting transformants showed that TALEN-induced double strand breaks led to the introduction of short deletions at the target site. Additional analysis revealed that each barley transformant contained a range of different mutations, indicating that mutations occurred independently in different cells.

Intragenesis and cisgenesis as alternatives to transgenic crop development.

One of the major concerns of the general public about transgenic crops relates to the mixing of genetic materials between species that cannot hybridize by natural means. To meet this concern, the two transformation concepts cisgenesis and intragenesis were developed as alternatives to transgenesis. Both concepts imply that plants must only be transformed with genetic material derived from the species itself or from closely related species capable of sexual hybridization. Furthermore, foreign sequences such as selection genes and vector-backbone sequences should be absent. Intragenesis differs from cisgenesis by allowing use of new gene combinations created by in vitro rearrangements of functional genetic elements. Several surveys show higher public acceptance of intragenic/cisgenic crops compared to transgenic crops. Thus, although the intragenic and cisgenic concepts were introduced internationally only 9 and 7 years ago, several different traits in a variety of crops have currently been modified according to these concepts. Five of these crops are now in field trials and two have pending applications for deregulation. Currently, intragenic/cisgenic plants are regulated as transgenic plants worldwide. However, as the gene pool exploited by intragenesis and cisgenesis are identical to the gene pool available for conventional breeding, less comprehensive regulatory measures are expected. The regulation of intragenic/cisgenic crops is presently under evaluation in the EU and in the US regulators are considering if a subgroup of these crops should be exempted from regulation. It is accordingly possible that the intragenic/cisgenic route will be of major significance for future plant breeding.

Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules.

Agrobacterium-mediated transformation of in vitro cultured barley ovules is an attractive alternative to well-established barley transformation methods of immature embryos. The ovule culture system can be used for transformation with and without selection and has successfully been used to transform cultivars other than Golden Promise indicating minor genotype dependency. The method allows for the rapid and direct generation of high-quality transgenic plants where the transgenes are stably expressed and show Mendelian inheritance in subsequent generations.

Transformation of different barley (Hordeum vulgare L.) cultivars by Agrobacterium tumefaciens infection of in vitro cultured ovules.

Most cultivars of higher plants display poor regeneration capacity of explants due to yet unknown genotypic determined mechanisms. This implies that technologies such as transformation often are restricted to model cultivars with good tissue characteristics. In the present paper, we add further evidence to our previous hypothesis that regeneration from young barley embryos derived from in vitro-cultured ovules is genotype independent. We investigated the ovule culture ability of four cultivars Femina, Salome, Corniche and Alexis, known to have poor response in other types of tissue culture, and compared that to the data for the model cultivar, Golden Promise. Subsequently, we analyzed the transformation efficiencies of the four cultivars using the protocol for Agrobacterium infection of ovules, previously developed for Golden Promise. Agrobacterium tumefaciens strain AGL0, carrying the binary vector pVec8-GFP harboring a hygromycin resistance gene and the green fluorescence protein (GFP) gene, was used for transformation. The results strongly indicate that the tissue culture response level in ovule culture is genotype independent. However, we did observe differences between cultivars with respect to frequencies of GFP-expressing embryos and frequencies of regeneration from the GFP-expressing embryos under hygromycin selection. The final frequencies of transformed plants per ovule were lower for the four cultivars than that for Golden Promise but the differences were not statistically significant. We conclude that ovule culture transformation can be used successfully to transform cultivars other than Golden Promise. Similar to that observed for Golden Promise, the ovule culture technique allows for the rapid and direct generation of high quality transgenic plants.

Transformation of barley (Hordeum vulgare L.) by Agrobacterium tumefaciens infection of in vitro cultured ovules.

We report on a novel transformation procedure for barley by Agrobacterium infection of in vitro cultured ovules. Ovules of the cultivar Golden Promise were isolated a few hours after pollination and infected with the Agrobacterium tumefaciens strain AGL0 carrying the binary vector pVec8-GFP. The vector harboured a hygromycin resistance gene and the green fluorescence protein (GFP) gene. GFP-expressing embryos were isolated from the ovules, regenerated to plants and investigated by Southern blot analysis. Transformation frequencies amounted to 3.1% with hygromycin selection and 0.8% without selection. Mendelian inheritance and stable expression of the GFP gene was confirmed in 18 independent lines over two generations. We conclude that the described technique allows for the rapid and direct generation of high quality transgenic plants.